The long-term goals of my laboratory are to understand the cellular and molecular mechanisms that underlie gamete interactions and cell-cell fusion during fertilization. We use the unicellular, biflagellated alga Chlamydomonas for our studies, an organism whose fertilization is highly amenable to cell biological, biochemical, molecular biological, and genetic approaches. Importantly, during Chlamydomonas fertilization, the interacting male and female gametes undergo the cell biological events that characterize fertilization in almost all animals. Understanding gamete fusion is needed to inform future studies on human reproduction. In addition, gamete fusion is essential for insect transmission of several devastating human diseases caused by parasitic protozoa, including the organisms that cause malaria and sleeping sickness. Knowing the molecular mechanisms of gamete fusion in parasitic protozoa would be an important advance in efforts to reduce the impact of the diseases they cause. Unfortunately, in spite of the many different fertilization systems being studied, our molecular understanding of gamete fusion in any organism remains rudimentary. The objectives of this proposal are to characterize the cellular and molecular mechanisms of gamete fusion in Chlamydomonas, focusing on two gamete fusion proteins, Fus1 a female gamete-specific protein, and a new male gamete-specific protein we discovered in this funding period, FusM. Both are localized at the sites of fusion and essential for fusion. Unlike all other gamete fusion proteins identified to date in any organism, FusM is widely conserved. Although not found in bilaterian animals, FusM family members are present in simple animals, higher plants, and pathogenic protozoa. In collaborative efforts using targeted gene disruption in Plasmodium berghei, a rodent malaria parasite, we discovered that the Plasmodium FusM is essential for gamete fusion. Thus, our studies on Chlamydomonas have revealed a conserved mechanism for gamete fusion. Now that we have a protein on each Chlamydomonas gamete that is essential for gamete fusion, we are in a unique position to make new insights into fundamental cellular and molecular mechanisms of gamete fusion. We propose to identify the binding partners of Fus1 and FusM, to determine the molecular functions of the proteins in gamete membrane adhesion and fusion, to investigate the molecular mechanisms of their localization, and to characterize their molecular properties before, during, and after fusion.